搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

The pathogenesis of malarial anemia is multifactorial,and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller,K.L.,J.C. Schooley,K.L. Smith,B. Kullgren,L.J. Mahlmann,and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap,G.S.,and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients,MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon,which are known antagonists of hematopoiesis,even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production,and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia,improved erythroid progenitor development,and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications. View Publication

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis,even if initial gene transfer efficiency is low. Moreover,selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However,a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore,understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly,not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection,thus underscoring the potential value of MGMT P140K selection for clinical gene therapy. View Publication

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However,this is a slow and inefficient process,depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover,once cell reprogramming is accomplished,these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However,no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus,which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes,deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore,interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus,this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. View Publication

Many drug-resistant tumors and cancer stem cells (CSC) express elevated levels of CD44 receptor,a cellular glycoprotein binding hyaluronic acid (HA). Here,we report the synthesis of nanogel-drug conjugates based on membranotropic cholesteryl-HA (CHA) for efficient targeting and suppression of drug-resistant tumors. These conjugates significantly increased the bioavailability of poorly soluble drugs with previously reported activity against CSC,such as etoposide,salinomycin,and curcumin. The small nanogel particles (diameter 20-40 nm) with a hydrophobic core and high drug loads (up to 20%) formed after ultrasonication and demonstrated a sustained drug release following the hydrolysis of biodegradable ester linkage. Importantly,CHA-drug nanogels demonstrated 2-7 times higher cytotoxicity in CD44-expressing drug-resistant human breast and pancreatic adenocarcinoma cells compared to that of free drugs and nonmodified HA-drug conjugates. These nanogels were efficiently internalized via CD44 receptor-mediated endocytosis and simultaneous interaction with the cancer cell membrane. Anchoring by cholesterol moieties in the cellular membrane after nanogel unfolding evidently caused more efficient drug accumulation in cancer cells compared to that in nonmodified HA-drug conjugates. CHA-drug nanogels were able to penetrate multicellular cancer spheroids and displayed a higher cytotoxic effect in the system modeling tumor environment than both free drugs and HA-drug conjugates. In conclusion,the proposed design of nanogel-drug conjugates allowed us to significantly enhance drug bioavailability,cancer cell targeting,and the treatment efficacy against drug-resistant cancer cells and multicellular spheroids. View Publication

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly,laborious,time-consuming,complex,and require skilled personnel. Hence,utilisation of most diagnostics for AAT is impracticable in rural areas,where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection,without relying on expensive equipment,would facilitate AAT detection. In turn,early management,would reduce disease incidence and severity. Today,several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context,we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T . congolense infections,which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here,originated from a past study. Briefly,the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T . congolense glycosomal fructose-1,6-bisphosphate aldolase ( Tco ALD) was discovered as the cognate Ag of Nb474H. In this study,splenocytes were harvested from a mouse immunized with recombinant Tco ALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on Tco ALD retrieved a lone binder,designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native Tco ALD released during infection by T . congolense parasites. Hitherto,development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) “hybrid” sandwich technology offers prospects for exploration,using the unique specificity of Nb as a key determinant in Ag capturing,while using the versatility of monoclonal Ab to adapt to various detection conditions. View Publication

Clinical translation of tissue-engineered advanced therapeutic medicinal products is hindered by a lack of patient-dependent and independent in-process biological quality controls that are reflective of in vivo outcomes. Recent insights into the mechanism of native bone repair highlight a robust path dependence. Organoid-based bottom-up developmental engineering mimics this path-dependence to design personalized living implants scaffold-free,with in-build outcome predictability. Yet,adequate (noninvasive) quality metrics of engineered tissues are lacking. Moreover,insufficient insight into the role of donor variability and biological sex as influencing factors for the mechanism toward bone repair hinders the implementation of such protocols for personalized bone implants. Here,male and female bone-forming organoids were compared to non-bone-forming organoids regarding their extracellular matrix composition,transcriptome,and secreted proteome signatures to directly link in vivo outcomes to quality metrics. As a result,donor variability in bone-forming callus organoids pointed towards two distinct pathways to bone,through either a hypertrophic cartilage or a fibrocartilaginous template. The followed pathway was determined early,as a biological sex-dependent activation of distinct progenitor populations. Independent of donor or biological sex,a cartilage-to-bone transition was driven by a common panel of secreted factors that played a role in extracellular matrix remodeling,mineralization,and attraction of vasculature. Hence,the secreted proteome is a source of noninvasive biomarkers that report on biological potency and could be the missing link toward data-driven decision-making in organoid-based bone tissue engineering. Subject terms: Bone,Bone quality and biomechanics View Publication

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas,despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention,the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment,IgG levels recovered. The IgG antibodies from naturally-infected,vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected,unvaccinated counterparts. Furthermore,peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response,in the form of increased and expanded IgG production and host response through neutrophil degranulation. View Publication

Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development,but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak,we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs,and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population,which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting,dissociation and re-plating reestablishes an epithelial phenotype. View Publication

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them,fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However,downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2,an inhibitor of protein kinase C (PKC),GF109203X (GFX),increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β),suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator,phorbol 12-myristate 13-acetate whereas Gö6976,a selective inhibitor of PKCα,β,and γ isoforms could not counteract the effect of PMA. Intriguingly,functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ,PKCε,and ζ,the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ,and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT,mitogen-activated protein kinase/ERK-1/2 kinase (MEK),PKC/ERK-1/2 kinase,and PKC/GSK-3β. Addition of GFX with a MEK inhibitor,U0126,in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells. View Publication

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4,MEF2C,and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However,GMT alone appears insufficient in human fibroblasts,at least in vitro. Here,we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells,fetal heart,and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming,including sarcomere formation,calcium transients,and action potentials,although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore,we found that transforming growth factor β signaling was important for,and improved the efficiency of,human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage,and lay the foundation for future refinements in vitro and in vivo. textcopyright 2013 The Authors. View Publication

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors,particularly radial glia (RG),are rare and are defined by a combination of intracellular markers,position and morphology. To circumvent these problems,we developed Fixed and Recovered Intact Single-cell RNA (FRISCR),a method for profiling the transcriptomes of individual fixed,stained and sorted cells. Using FRISCR,we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB,and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles,an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers. View Publication